DESCRIPTION: (Applicant's abstract) Single nucleotide variation occurs throughout the human genome and contributes to the remarkable phenotypic variation present in the human race. For studying this variation, particularly with an aim to identify genes involved in common, multigenic diseases, very large numbers of single nucleotide polymorphisms (SNPs) must be identified and mapped in the human genome. This proposal aims to develop a mismatch enrichment method that will increase the efficiency with which SNPs can be discovered in any complex genome. In this method, 300-base pair restriction enzyme-digested fragments representing about 5% of the human genome will be isolated from DNA of four unrelated individuals. Heteroduplexes will be formed between the different alleles in this mixture of DNA by denaturation and rehybridization. Those fragments containing a mismatch, estimated to be about one out of seven fragments, will be purified from the non-mismatched fragments by binding to MutS protein, which is involved in mismatch repair in E. coli. The DNA fragment population enriched for mismatches will be cloned into a plasmid in E. coli under conditions that maintain both alleles in each resulting bacterial colony. Four-color fluorescent sequencing will be performed from each colony, such that the DNA sequence of the 300 bp fragment, as well as the position and composition of the two alleles, will be determined. For those DNA segments containing a SNP, PCR primers will be designed so that the fragment can be specifically amplified from total human genomic DNA. During the two year project efficacy of this method will be tested by discovering and sequencing approximately 1,000 such SNPs, and analyzing about 100 of these further by determining their map positions in the human genome and their allele frequencies in ten unrelated people.